Identification of a novel protein in the genome sequences of Leptospira interrogans with the ability to interact with host's components.

BACKGROUND: Leptospirosis is an infectious disease that affects humans and animals worldwide. The etiological agents of this disease are the pathogenic species of the genus Leptospira. The mechanisms involved in the leptospiral pathogenesis are not full understood. The elucidation of novel mediators of host-pathogen interaction is important in the detection of virulence factors involved in the pathogenesis of leptospirosis. OBJECTIVE: This work focused on identification and characterization of a hypothetical protein of Leptospira encoded by the gene LIC10920. METHODS: The protein of unknown function was predicted to be surface exposed. Therefore, the LIC10920 gene was cloned and the protein expressed in Escherichia coli BL21 (DE3) Star pLysS strain. The recombinant protein was purified by metal affinity chromatography and evaluated with leptospirosis human serum samples. The interaction with host components was also performed. RESULTS: The recombinant protein was recognized by antibodies present in leptopsirosis human serum, suggesting its expression during infection. Immunofluorescence and intact bacteria assays indicated that the bacterial protein is surface-exposed. The recombinant protein interacted with human laminin, in a dose-dependent and saturable manner and was named Lsa24.9, for Leptospiral surface adhesin, followed by its molecular mass. Lsa24.9 also binds plasminogen (PLG) in a dose-dependent and saturable fashion, fulfilling receptor ligand interaction. Moreover, Lsa24.9 has the ability to acquire PLG from normal human serum, exhibiting similar profile as observed with the human purified component. PLG bound Lsa24.9 was able of generating plasmin, which could increase the proteolytic power of the bacteria. CONCLUSIONS: This novel leptospiral protein may function as an adhesin at the colonization steps and may help the invasion process by plasmin generation at the bacterial cell surface.

Identification of a novel protein in the genome sequences of Leptospira interrogans with the ability to interact with host's components

Background Leptospirosis is an infectious disease that affects humans and animals worldwide. The etiological agents of this disease are the pathogenic species of the genus Leptospira. The mechanisms involved in the leptospiral pathogenesis are not full understood. The elucidation of novel mediators of host-pathogen interaction is important in the detection of virulence factors involved in the pathogenesis of leptospirosis. Objective This work focused on identification and characterization of a hypothetical protein of Leptospira encoded by the gene LIC10920. Methods The protein of unknown function was predicted to be surface exposed. Therefore, the LIC10920 gene was cloned and the protein expressed in Escherichia coli BL21 (DE3) Star pLysS strain. The recombinant protein was purified by metal affinity chromatography and evaluated with leptospirosis human serum samples. The interaction with host components was also performed. Results The recombinant protein was recognized by antibodies present in leptopsirosis human serum, suggesting its expression during infection. Immunofluorescence and intact bacteria assays indicated that the bacterial protein is surface-exposed. The recombinant protein interacted with human laminin, in a dose-dependent and saturable manner and was named Lsa24.9, for Leptospiral surface adhesin, followed by its molecular mass. Lsa24.9 also binds plasminogen (PLG) in a dose-dependent and saturable fashion, fulfilling receptor ligand interaction. Moreover, Lsa24.9 has the ability to acquire PLG from normal human serum, exhibiting similar profile as observed with the human purified component. PLG bound Lsa24.9 was able of generating plasmin, which could increase the proteolytic power of the bacteria. Conclusions This novel leptospiral protein may function as an adhesin at the colonization steps and may help the invasion process by plasmin generation at the bacterial cell surface.

Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira.

Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the ß-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.

Gene silencing based on RNA-guided catalytically inactive Cas9 (dCas9): a new tool for genetic engineering in Leptospira

Leptospirosis is a worldwide zoonosis caused by pathogenic bacteria of the genus Leptospira, which also includes free-living saprophyte strains. Many aspects of leptospiral basic biology and virulence mechanisms remain unexplored mainly due to the lack of effective genetic tools available for these bacteria. Recently, the type II CRISPR/Cas system from Streptococcus pyogenes has been widely used as an efficient genome engineering tool in bacteria by inducing double-strand breaks (DSBs) in the desired genomic targets caused by an RNA-guided DNA endonuclease called Cas9, and the DSB repair associated machinery. In the present work, plasmids expressing heterologous S. pyogenes Cas9 in L. biflexa cells were generated, and the enzyme could be expressed with no apparent toxicity to leptospiral cells. However, L. biflexa cells were unable to repair RNA-guided Cas9-induced DSBs. Thus, we used a catalytically dead Cas9 (dCas9) to obtain gene silencing rather than disruption, in a strategy called CRISPR interference (CRISPRi). We demonstrated complete gene silencing in L. biflexa cells when both dCas9 and single-guide RNA (sgRNA) targeting the coding strand of the ß-galactosidase gene were expressed simultaneously. Furthermore, when the system was applied for silencing the dnaK gene, no colonies were recovered, indicating that DnaK protein is essential in Leptospira. In addition, flagellar motor switch FliG gene silencing resulted in reduced bacterial motility. To the best of our knowledge, this is the first work applying the CRISPRi system in Leptospira and spirochetes in general, expanding the tools available for understanding leptospiral biology.

Identification and Perceived Intensity of Facial Expressions of Emotion in Bipolar Disorder and Major Depression.

OBJECTIVE: This study aimed to examine the accuracy in identifying facial expressions and the perceived intensity of emotions of happiness, sadness, disgust, anger, fear, surprise in bipolar disorder (BD), and major depressive disorder (MDD). METHOD: Ninety-three adult participants (n = 30 BD, 80% female; n = 18 MDD, 72.2% female; n = 45 C, 46.7% female) completed a facial expression task. Stimuli in the task were first presented for 200 ms, followed by 500 ms, and 1000 ms. Participants were asked to identify the emotion expressed by each face and judge its intensity on a Likert scale. Accuracy and perceived intensity of expressions corresponding to each emotion were compared between groups using a repeated measures ANCOVA, with length of stimulus presentation as a within-subjects variable. RESULTS: Expressions of sadness were rated more intensely by patients with BD, and expressions of anger by those with MDD, than control participants. Subjects with BD were less accurate than control participants in identifying expressions of disgust. An interaction effect was also identified for the detection of sadness, whereby patients with BD were significantly less accurate when expressions were shown for 200 ms. CONCLUSIONS: The increased intensity with which emotions were perceived by patients with BD and MDD has important repercussions for patient functioning and clinical practice. A tendency to overestimate the intensity of certain facial expressions in mood disorders may lead patients to interpret social cues erroneously and engage in dysfunctional behaviors and cognitive patterns. Future studies should focus on this variable in addition to the accuracy of emotion identification.

Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant.

Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigA(C)) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigA(C), either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigA(C) or LigA(C) coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen.

Evaluation of the elastinolytic activity and protective effect of Leptallo I, a protein composed by metalloprotease and FA5/8C domains, from Leptospira interrogans Copenhageni.

Leptospirosis is a re-emergent zoonosis, caused by pathogenic spirochetes from the genus Lepstospira. To date, there is no protein described to be involved in leptospiral hemorrhagic manifestations, although several proteases have been reported for other bacterial infections. In this study we identified 12 putative metalloproteases from the genome of Leptospira interrogans, and characterized for the first time a putative metalloprotease, here named Leptallo I, as a potential Zn(2+) dependent glycylglycine protease belonging to the M23 metalloendopeptidase family. The native protein was detected in extracts from several pathogenic Leptospira species and further shown to be secreted to the culture medium. We expressed the recombinant protein and its C-terminal fragment containing the metalloprotease domain, and both presented regular secondary structures. The sera of humans with leptospirosis were able to recognize rLeptallo I, indicating that the native protein is expressed and presented to the immune system during infection. The recombinant proteins displayed a significant, though relatively low, elastinolytic activity, and the challenge of hamsters immunized with rLeptallo I conferred 33% protection, suggesting a significant importance of this protein in the pathogenesis. The elastinolytic activity may be important for leptospires-host interaction, because elastin constitutes a significant proportion of total lung and blood vessel proteins.

The potential for hydrocarbon biodegradation and production of extracellular polymeric substances by aerobic bacteria isolated from a Brazilian petroleum reservoir.

Extracellular polymeric substances (EPS) can contribute to the cellular degradation of hydrocarbons and have a huge potential for application in biotechnological processes, such as bioremediation and microbial enhanced oil recovery (MEOR). Four bacterial strains from a Brazilian petroleum reservoir were investigated for EPS production, emulsification ability and biodegradation activity when hydrocarbons were supplied as substrates for microbial growth. Two strains of Bacillus species had the highest EPS production when phenanthrene and n-octadecane were offered as carbon sources, either individually or in a mixture. While Pseudomonas sp. and Dietzia sp., the other two evaluated strains, had the highest hydrocarbon biodegradation indices, EPS production was not detected. Low EPS production may not necessarily be indicative of an absence of emulsifier activity, as indicated by the results of a surface tension reduction assay and emulsification indices for the strain of Dietzia sp. The combined results gathered in this work suggest that a microbial consortium consisting of bacteria with interdependent metabolisms could thrive in petroleum reservoirs, thus overcoming the limitations imposed on each individual species by the harsh conditions found in such environments.

Comparison of agglutinating and growth inhibition antibodies levels in sows immunized with two experimental inactivated monovalent vaccines of Canicola serovar: whole culture with aluminum hydroxide and lipopolysaccharide subunit with lipid A / Comparação dos níveis de anticorpos pós-vacinais, aglutinantes e inibidores do crescimento de leptospiras em fêmeas suínas imunizadas com duas vacinas inativadas experimentais monovalentes do sorovar Canicola: cultivo total inativado com hidróxido de alumínio e subunidade de lipopolissacarídios com monofosforil lipídio A

It was compared the antibody response of sows immunized with two experimental vaccines produced with L.interrogans, serovar Canicola, strain LO-4, isolated in Brazil.One of the vaccines was the usual bacterin (whole culture inactivated with phenol and adjuvanted with aluminum hydroxide -WC-AlOH3) and the other one was a subunit vaccine produced with a lipopolysaccharide (LPS) fraction extracted from the bacteria outer envelop and with the lipid A, also extracted from the leptospira wall as adjuvant (LPS-MPLA). Experiment was as follows: group 1 (n = 11), not immunized control, group 2, (n = 11): two immunization with 30 days interval of LPS-MPLA vaccine and group 3 (n = 11): two immunization with 30 days interval of WC-AlOH3 vaccine All three groups were simultaneously immunized, independently of pregnancy stage. Both agglutinin and neutralizing post vaccination antibodies levels were measured respectively by the microscopic sera agglutination with live antigens test (MAT) and the in vitro leptospira growth inhibition test (GIT). Sera collections were performed each 30 days during four months after the first vaccination. Non vaccinated control group animals presented no agglutinating antibodies against Canicola serovar during the whole experiment. At 32 and 68 post vaccination days the agglutinating antibodies levels of group 2 (LPS-MPLA) were significantly higher than the observed in group 3 (WC AlOH3), respectively, p = 0.013 and p = 0.031. The differences observed in the growth inhibition antibodies titers of the two vaccines tested were not significant (p > 0.05). Despite the peak of post-vaccination agglutinins have been registered at 68 days after first immunization, higher levels growth inhibition antibodies were detected at 30 days of first vaccination. Subunit vaccine presented the same immunogenic capacity for the production of neutralizing antibodies as the whole culture one.

Foi comparada a resposta imune de fêmeas suínas adultas imunizadas contra a leptospirose, com vacinas monovalentes produzidas com L.interrogans, sorovar Canicola estirpe LO4, isolada no Brasil. A vacina foi empregada em duas formas: cultura de bactérias totais inativada e acrescida do adjuvante de hidróxido de alumínio (WC-AlOH3) e a do tipo de subunidade constituída apenas por uma fração de lipopolisacarídios (LPS) extraídos do envelope externo da bactéria tendo como adjuvante o monofosforil lipídio A, também extraído da parede da leptospira (LPS-MPLA). O delineamento experimental incluiu: grupo 1 (n = 11): controle não imunizado; grupo 2 (n = 11): imunizado com duas aplicações em intervalo de 30 dias da vacina LPS MFLA; Grupo 3 (n = 11): imunizado com duas aplicações em intervalo de 30 dias da vacina WC-AlOH3. Todos os grupos foram imunizados simultaneamente sem ser considerado o estágio de gestação dos animais. Os níveis de anticorpos pós-vacinais, aglutinantes e neutralizantes foram avaliados, respectivamente, pelos testes de soroaglutinação microscópica com antígenos vivos (SAM) e o de inibição do crescimento de leptospiras in vitro (ICLIV). O monitoramento sorológico foi efetuado a cada 30 dias durante quatro meses após aplicação da primeira dose da vacina. Os animais do grupo controle, não vacinados, não apresentaram anticorpos aglutinantes para o sorovar Canicola durante todo o período experimental. Aos 32 e 68 dias da primo-vacinação, os níveis de anticorpos aglutinantes do grupo 2 (LPS-MPLA) foram significativamente superiores aos observados no grupo 3 (WC AlOH3), respectivamente p = 0,013 e p = 0,031. As diferenças observadas nos níveis de anticorpos inibidores do crescimento de leptospiras in vitro, induzidos pelas duas vacinas, não foram significativas (p > 0,05). A despeito do pico de anticorpos aglutinantes pós-vacinais ter sido registrado aos 68 dias da primeira imunização, os níveis mais elevados de anticorpos inibidores do crescimento de leptospiras já foram observados aos 30 dias da primo-vacinação. A vacina de subunidade apresentou a mesma capacidade de indução de anticorpos neutralizantes que a vacina de bactérias totais.

COMPARISON OF AGGLUTINATING AND GROWTH INHIBITION ANTIBODIES LEVELS IN SOWS IMMUNIZED WITH TWO EXPERIMENTAL INACTIVATED MONOVALENT VACCINES OF CANICOLA SEROVAR: WHOLE CULTURE WITH ALUMINUM HYDROXIDE AND LIPOPOLYSACCHARIDE SUBUNIT WITH LIPID A

ABSTRACT It was compared the antibody response of sows immunized with two experimental vaccines produced with L.interrogans, serovar Canicola, strain LO-4, isolated in Brazil.One of the vaccines was the usual bacterin (whole culture inactivated with phenol and adjuvanted with aluminum hydroxide -WC-AlOH3) and the other one was a subunit vaccine produced with a lipopolysaccharide (LPS) fraction extracted from the bacteria outer envelop and with the lipid A, also extracted from the leptospira wall as adjuvant (LPS-MPLA). Experiment was as follows: group 1 (n = 11), not immunized control, group 2, (n = 11): two immunization with 30 days interval of LPS-MPLA vaccine and group 3 (n = 11): two immunization with 30 days interval of WC-AlOH3 vaccine All three groups were simultaneously immunized, independently of pregnancy stage. Both agglutinin and neutralizing post vaccination antibodies levels were measured respectively by the microscopic sera agglutination with live antigens test (MAT) and the in vitro leptospira growth inhibition test (GIT). Sera collections were performed each 30 days during four months after the first vaccination. Non vaccinated control group animals presented no agglutinating antibodies against Canicola serovar during the whole experiment. At 32 and 68 post vaccination days the agglutinating antibodies levels of group 2 (LPS-MPLA) were significantly higher than the observed in group 3 (WC AlOH3), respectively, p = 0.013 and p = 0.031. The differences observed in the growth inhibition antibodies titers of the two vaccines tested were not significant (p > 0.05). Despite the peak of post-vaccination agglutinins have been registered at 68 days after first immunization, higher levels growth inhibition antibodies were detected at 30 days of first vaccination. Subunit vaccine presented the same immunogenic capacity for the production of neutralizing antibodies as the whole culture one.

RESUMO Foi comparada a resposta imune de fêmeas suínas adultas imunizadas contra a leptospirose, com vacinas monovalentes produzidas com L.interrogans, sorovar Canicola estirpe LO4, isolada no Brasil. A vacina foi empregada em duas formas: cultura de bactérias totais inativada e acrescida do adjuvante de hidróxido de alumínio (WC-AlOH3) e a do tipo de subunidade constituída apenas por uma fração de lipopolisacarídios (LPS) extraídos do envelope externo da bactéria tendo como adjuvante o monofosforil lipídio A, também extraído da parede da leptospira (LPS-MPLA). O delineamento experimental incluiu: grupo 1 (n = 11): controle não imunizado; grupo 2 (n = 11): imunizado com duas aplicações em intervalo de 30 dias da vacina LPS MFLA; Grupo 3 (n = 11): imunizado com duas aplicações em intervalo de 30 dias da vacina WC-AlOH3. Todos os grupos foram imunizados simultaneamente sem ser considerado o estágio de gestação dos animais. Os níveis de anticorpos pós-vacinais, aglutinantes e neutralizantes foram avaliados, respectivamente, pelos testes de soroaglutinação microscópica com antígenos vivos (SAM) e o de inibição do crescimento de leptospiras in vitro (ICLIV). O monitoramento sorológico foi efetuado a cada 30 dias durante quatro meses após aplicação da primeira dose da vacina. Os animais do grupo controle, não vacinados, não apresentaram anticorpos aglutinantes para o sorovar Canicola durante todo o período experimental. Aos 32 e 68 dias da primo-vacinação, os níveis de anticorpos aglutinantes do grupo 2 (LPS-MPLA) foram significativamente superiores aos observados no grupo 3 (WC AlOH3), respectivamente p = 0,013 e p = 0,031. As diferenças observadas nos níveis de anticorpos inibidores do crescimento de leptospiras in vitro, induzidos pelas duas vacinas, não foram significativas (p > 0,05). A despeito do pico de anticorpos aglutinantes pós-vacinais ter sido registrado aos 68 dias da primeira imunização, os níveis mais elevados de anticorpos inibidores do crescimento de leptospiras já foram observados aos 30 dias da primo-vacinação. A vacina de subunidade apresentou a mesma capacidade de indução de anticorpos neutralizantes que a vacina de bactérias totais.

Anticorpos anti-Leptospira spp. em ovinos do município de Monte Negro, estado de Rondônia / Anti-Leptospira spp. antibodies in ovines from Monte Negro municipality, Rondonia state, Brazil

O presente estudo determinou a prevalência de anticorpos anti-Leptospira spp. em ovinos do Município de Monte Negro, RO. Foram examinados soros de 141 ovinos de raça, idade e sexo variados provenientes de 15 fazendas, pela técnica de Soroaglutinação Microscópica. Doze (80,0%) propriedades apresentaram pelo menos um animal reagente. Títulos de anticorpos iguais ou superiores a 100 foram detectados em 47 (33,3%) animais, e os sorovares mais frequentes foram Patoc (29,7%), Autumnalis (14,8%), Pyrogenes (10,6%), Australis (4,2%), Bratislava (4,2%), Hardjo (4,2%), Icterohaemorrhagiae (4,2%), Castellonis (2,1%) e Hebdomadis (2,1%). Em 11 (23,4%) soros não foi possível a determinação do provável sorovar envolvido na reação. Alerta-se também para a possibilidade de infecção no homem, tendo em vista as características regionais de fronteira agrícola amazônica.

The present study determined the prevalence of anti-Leptospira spp.antibodies in 141 ovines from 15 farms of the Monte Negro Municipality, Rondonia State, Brazil, by the microscopic agglutination test. Twelve (80.0%) farms presented at least 1 reactive animal. Antibodies titers of ? 100 were detected in 47 (33.3%) animals, the most frequent serovars being Patoc (29.7%), Autumnalis (14.8%), Pyrogenes (10.6%), Australis (4.2%), Bratislava (4.2%), Hardjo (4.2%), Icterohaemorrhagiae (4.2%), Castellonis (2.1%) and Hebdomadis (2.1%). In 11 (23.4%) sera it was not possible to determine the most frequent serovar involved. The results raise a warning as to the possibility of infection in the human being by Leptospira in light of the regional characteristics of the Amazon agricultural frontier.

Comparison of a PCR assay in whole blood and serum specimens for canine brucellosis diagnosis.

The performance of a serum PCR assay was compared with that of a blood PCR assay for the diagnosis of canine brucellosis caused by Brucella canis in 72 dogs. The dogs were classified into three groups (infected, non-infected and suspected brucellosis) according to the results of blood culture and serological tests. The sensitivities of blood PCR and serum PCR were, respectively, 97.14 per cent and 25.71 per cent. The specificities of both were 100 per cent. In the group of dogs with suspected brucellosis, three were positive by blood PCR and none was positive by serum PCR. Serum PCR showed little value for the direct diagnosis of canine brucellosis as the assay had low diagnostic sensitivity and fewer positive dogs were detected by this test than by blood culture, blood PCR, rapid slide agglutination test (RSAT) and RSAT with 2-mercaptoethanol.

Tuberculose em caprinos do semiárido brasileiro / Tuberculosis in goats from a semiarid region in Brazil

No período de janeiro a outubro de 2007, foram tuberculinizadas 1.866 fêmeas caprinas de diferentes raças e idades distribuídas em 84 propriedades da microrregião de Monteiro, semiárido da Paraíba. Nove cabras (0,48%) foram positivas, duas delas foram sacrificadas e, à necropsia foram observadas lesões sugestivas de tuberculose nos pulmões e no fígado. No exame histopatológico, foram evidenciados granulomas típicos. Foi procedido o cultivo e isolamento de micobactérias, sendo observado o crescimento do agente no meio de cultura de Stonebrink a partir de 36 dias de incubação, com um tempo médio de crescimento de 42 dias. Também foram encontrados resultados positivos na pesquisa de bacilos álcool-ácido resistentes (BAAR) pela coloração de Ziehl-Neelsen. O cultivo de Corynebacterium pseudotuberculosis foi negativo. O presente relato, com isolamento e visualização do agente, bem como verificação de lesões macro e microscópicas, é o primeiro caso de tuberculose confirmado em caprinos do semiárido brasileiro.

From January to October 2007, 1,866 female goats of different breeds and ages from 84 herds of the Monteiro microregion, in a semiarid area of Paraíba State, Brazil, were tuberculinized. Nine goats (0.48%) were positive and two of them were slaughtered, and at necropsy tuberculosis-like lesions were found in lungs and liver. At the histopathologic examination, typical tuberculosis granulomas were observed. Micobacteria culture and isolation were carried out, and there was growth of the agent on the Stonebrink medium culture from 36 days of inoculation with an average growth time of 42 days. Positive results were also found in the research of acid-fast bacilli by Ziehl-Neelsen staining. The culture of Corynebacterium pseudotuberculosis was negative. This report, with isolation and visualization of the agent, as well as verification of gross and microscopic lesions, is the first confirmed case of tuberculosis in goats of a semiarid region of Brazil.

Evaluation of the expression and protective potential of leptospiral sphingomyelinases

Leptospirosis is a zoonotic disease of global distribution, which affects both animals and humans. Pathogenic leptospires, the bacteria that cause this disease, require iron for their growth, and these spirochetes probably use their hemolysins, such as the sphingomyelinases, as a way to obtain this important nutrient from host red blood cells during infection. We expressed and purified the leptospiral sphingomyelinases Sph1, Sph2, Sph4, and SphH in a heterologous system. However, the recombinant proteins were not able to lyse sheep erythrocytes, despite having regular secondary structures. Transcripts for all sphingomyelinases tested were detected by RT-PCR analyses, but only Sph2 and SphH native proteins could be detected in Western blot assays using Leptospira whole extracts as well as in renal tubules of infected hamsters. Moreover, antibodies present in the serum of a human patient with laboratory-confirmed leptospirosis recognized Sph2, indicating that this sphingomyelinase is expressed and exposed to the immune system during infection in humans. However, in an animal challenge model, none of the sphingomyelinases tested conferred protection against leptospirosis.

ANTICORPOS ANTI-LEPTOSPIRA SPP. EM OVINOS DO MUNICÍPIO DE MONTE NEGRO, ESTADO DE RONDÔNIA

ABSTRACT The present study determined the prevalence of anti-Leptospira spp.antibodies in 141 ovines from 15 farms of the Monte Negro Municipality, Rondonia State, Brazil, by the microscopic agglutination test. Twelve (80.0%) farms presented at least 1 reactive animal. Antibodies titers of ? 100 were detected in 47 (33.3%) animals, the most frequent serovars being Patoc (29.7%), Autumnalis (14.8%), Pyrogenes (10.6%), Australis (4.2%), Bratislava (4.2%), Hardjo (4.2%), Icterohaemorrhagiae (4.2%), Castellonis (2.1%) and Hebdomadis (2.1%). In 11 (23.4%) sera it was not possible to determine the most frequent serovar involved. The results raise a warning as to the possibility of infection in the human being by Leptospira in light of the regional characteristics of the Amazon agricultural frontier.

RESUMO O presente estudo determinou a prevalência de anticorpos anti-Leptospira spp. em ovinos do Município de Monte Negro, RO. Foram examinados soros de 141 ovinos de raça, idade e sexo variados provenientes de 15 fazendas, pela técnica de Soroaglutinação Microscópica. Doze (80,0%) propriedades apresentaram pelo menos um animal reagente. Títulos de anticorpos iguais ou superiores a 100 foram detectados em 47 (33,3%) animais, e os sorovares mais frequentes foram Patoc (29,7%), Autumnalis (14,8%), Pyrogenes (10,6%), Australis (4,2%), Bratislava (4,2%), Hardjo (4,2%), Icterohaemorrhagiae (4,2%), Castellonis (2,1%) e Hebdomadis (2,1%). Em 11 (23,4%) soros não foi possível a determinação do provável sorovar envolvido na reação. Alerta-se também para a possibilidade de infecção no homem, tendo em vista as características regionais de fronteira agrícola amazônica.

TUBERCULOSE EM CAPRINOS DO SEMIÁRIDO BRASILEIRO

ABSTRACT From January to October 2007, 1,866 female goats of different breeds and ages from 84 herds of the Monteiro microregion, in a semiarid area of Paraíba State, Brazil, were tuberculinized. Nine goats (0.48%) were positive and two of them were slaughtered, and at necropsy tuberculosis-like lesions were found in lungs and liver. At the histopathologic examination, typical tuberculosis granulomas were observed. Micobacteria culture and isolation were carried out, and there was growth of the agent on the Stonebrink medium culture from 36 days of inoculation with an average growth time of 42 days. Positive results were also found in the research of acid-fast bacilli by Ziehl-Neelsen staining. The culture of Corynebacterium pseudotuberculosis was negative. This report, with isolation and visualization of the agent, as well as verification of gross and microscopic lesions, is the first confirmed case of tuberculosis in goats of a semiarid region of Brazil.

RESUMO No período de janeiro a outubro de 2007, foram tuberculinizadas 1.866 fêmeas caprinas de diferentes raças e idades distribuídas em 84 propriedades da microrregião de Monteiro, semiárido da Paraíba. Nove cabras (0,48%) foram positivas, duas delas foram sacrificadas e, à necropsia foram observadas lesões sugestivas de tuberculose nos pulmões e no fígado. No exame histopatológico, foram evidenciados granulomas típicos. Foi procedido o cultivo e isolamento de micobactérias, sendo observado o crescimento do agente no meio de cultura de Stonebrink a partir de 36 dias de incubação, com um tempo médio de crescimento de 42 dias. Também foram encontrados resultados positivos na pesquisa de bacilos álcool-ácido resistentes (BAAR) pela coloração de Ziehl-Neelsen. O cultivo de Corynebacterium pseudotuberculosis foi negativo. O presente relato, com isolamento e visualização do agente, bem como verificação de lesões macro e microscópicas, é o primeiro caso de tuberculose confirmado em caprinos do semiárido brasileiro.

Fatores de risco para a leptospirose em fêmeas bovinas em idade reprodutiva no Estado de São Paulo / Risk factors to leptospirosis in cows in São Paulo state, Brazil

Risk factors associated to leptospirosis were identified in cows from São Paulo State. The State was divided into seven productive circuits from which 8,216 cows older than 24 months of age from 1,021 herds were sampled. For serological diagnosis of Leptospira spp. infection, the microscopic agglutination test (MAT) was carried out. Of the investigated herds, 718 (71.3%; 95% CI = 68.5% - 74.0%) presented at least one reactant animal at MAT to any serovar. Serovar Hardjo was the most prevalent, with 55.2% (95% CI = 51.4% - 58.9%) of the positive herds. Herd size, animal purchase, share pasture, presence of ovine and swine, and utilization of artificial insemination were identified as risk factors. Utilization of maternity pens was a protective factor against leptospirosis.

In vitro degradation of linamarin by microorganisms isolated from cassava wastewater treatment lagoons

This study aimed at isolating and characterizing of microorganisms able to use linamarin as sole carbon source. Thirty one microbial strains were isolated from manipueira, a liquid effluent of cassava processing factories. Among these strains, Bacillus licheniformis (isolate 2_2) and Rhodotorulla glutinis (isolate L1) were able to degrade 71 percent and 95 percent of added linamarin, respectively, within 7 days, showing high biodegradation activity and great potential for detoxification of cassava processing wastewaters.

Fatores de risco para a leptospirose em fêmeas bovinas em idade reprodutiva no Estado de São Paulo / Risk factors to leptospirosis in cows in São Paulo state, Brazil

Risk factors associated to leptospirosis were identified in cows from São Paulo State. The State was divided into seven productive circuits from which 8,216 cows older than 24 months of age from 1,021 herds were sampled. For serological diagnosis of Leptospira spp. infection, the microscopic agglutination test (MAT) was carried out. Of the investigated herds, 718 (71.3%; 95% CI = 68.5% - 74.0%) presented at least one reactant animal at MAT to any serovar. Serovar Hardjo was the most prevalent, with 55.2% (95% CI = 51.4% - 58.9%) of the positive herds. Herd size, animal purchase, share pasture, presence of ovine and swine, and utilization of artificial insemination were identified as risk factors. Utilization of maternity pens was a protective factor against leptospirosis. (AU)

Situação epidemiológica da brucelose bovina no Estado do Espírito Santo / Epidemiological situation of bovine brucellosis in the State of Espírito Santo, Brazil

Realizou-se um estudo para caracterizar a situação epidemiológica da brucelose bovina no Estado do Espírito Santo. O Estado foi dividido em dois circuitos produtores. Em cada circuito foram amostradas aleatoriamente cerca de 300 propriedades e, dentro dessas, foi escolhido de forma aleatória um número pré-estabelecido de animais, dos quais foi obtida uma amostra de sangue. No total, foram amostrados 5.351 animais, provenientes de 622 propriedades. Em cada propriedade amostrada foi aplicado um questionário epidemiológico para verificar o tipo de exploração e as práticas de criação e sanitárias que poderiam estar associadas ao risco de infecção pela doença. O protocolo de testes utilizado foi o da triagem com o teste do antígeno acidificado tamponado e o reteste dos positivos com o teste do 2-mercaptoetanol. O rebanho foi considerado positivo quando pelo menos um animal foi reagente às duas provas sorológicas. Para o Estado, as prevalências de focos e de animais infectados foram, respectivamente, de 9,0 por cento [7,0-11,6 por cento] e 3,5 por cento [1,9-6,4 por cento]. Para os circuitos, as prevalências de focos e de animais infectados foram, respectivamente, de: circuito 1, 6,8 por cento [4,5-10,2 por cento] e 3,4 por cento [1,3-8,6 por cento]; circuito 2, 10,9 por cento [7,9 por cento-14,8 por cento] e 3,7 por cento [2,1-6,3 por cento]. Os fatores de risco (odds ratio, OR) associados à condição de foco foram: utilização de inseminação artificial (OR = 7,05 [2,51-19,82]) e confinamento/semiconfinamento dos animais (OR = 2,98 [1,22-7,26]). A vacinação de fêmeas entre três e oito meses de idade foi um fator protetor (OR = 0,03 [0,01-0,1]).

A study to characterize the epidemiological status of brucellosis was carried out in the State of Espírito Santo. The State was divided in two regions. Three hundred herds were randomly sampled in each region and a pre-established number of animals were sampled in each of these herds. A total of 5,351 serum samples from 622 herds were collected. In each herd, it was applied an epidemiological questionnaire focused on herd traits as well as husbandry and sanitary practices that could be associated with the risk of infection. The serum samples were screened for antibodies against Brucella spp. by the Rose-Bengal Test (RBT), and all positive sera were re-tested by the 2-mercaptoethanol test (2-ME). The herd was considered positive if at least one animal was positive on both RBT and 2-ME tests. The prevalence of infected herds and animals in the State were, respectively, 9.0 percent [7.0-11.6 percent] and 3.5 percent [1.9-6.4 percent]. The prevalence of infected herds and animals in the regions were, respectively: region 1, 6.8 percent [4.5-10.2 percent] and 3.4 percent [1.3-8.6 percent]; and region 2, 10.9 percent [7.9-14.8 percent] and 3.7 percent [2.1-6.3 percent]. The risk factors (odds ratio, OR) associated with the presence of the infection were: use of artificial insemination (OR = 7.05 [2.51-19.82]) and intensive/semi-intensive management systems (OR = 2.98 [1.22-7.26]). Vaccination of heifers from three to eight months of age was a protective factor (OR = 0.03 [0.01-0.1]).